The invention relates, in general, to methods and devices useful for analytical testing and, in particular, to methods and devices for flow-through optical assay.
An optical assay device is a device used to detect an analyte such as an antigen. These devices may carry an optically active test member to which a sample is applied for determining the presence or amount of an analyte of interest.
It is desirable in an assay device for the optically active test member to be extremely sensitive to the existence of an analyte, and for the assay performance time, i.e., incubation time, to be as short as possible. This is accomplished in flow-through optical assay devices by maximizing the sample volume which is brought in contact with an analyte specific receptive material or the test member and controlling the flow characteristics of the sample through the optical member.
Although the sample will flow through the optical member without external assistance, the flow characteristics of the sample across the optical member and through channels within the optical member or around the optical member can be modified by the use of absorbent materials. Absorbent material allows for wicking which acts to draw fluid from the surface that the adsorbent material is in contact with which can cause fluid to be drawn across the layers of the optical member and through the channels within the optical member or around the optical member. The absorbent material also provides drying of the optical member when contacted with the optical stack. This drying helps to distinguish the signal produced by the optical member.
U.S. Pat. No. 5,418,136 (Miller et al.) describes a blotting device and blotting method which uses an optically reactive surface as the receptor for samples and reagents related to the particular assay being performed. The device contains an optically reactive layer supported on a pedestal of the device in order to allow placement of various solutions, e.g. sample, washing reagents, substrate, directly onto the reactive layer""s top surface. The solutions are removed by blotting the reactive surface with an absorbent material by physically pressing the absorbent material onto the reactive surface.
These optical assay devices require that the user apply a discrete volume of sample (approximately 25-30 xcexcL) on the surface and that the incubation times be controlled by user intervention. Sample incubates on the surface in a static mode as the surface is solid and impermeable. The drying process also requires user interaction to bring the adsorbent material into contact with the solid optical test surface from above the test surface. While the solid surface optical assays are extremely sensitive, an improvement in sensitivity can be gained by using all of the available sample (dependent on sample processing but generally greater than 200 xcexcL). In many testing sites, the requirement for user intervention in timing and drying the optical test device is inconvenient and not cost effective.
The prior art also includes assay devices that allow for sample flow through the surface of a porous material or across a tortuous path material. Detection is based on the generation of a calorimetric signal through the use of a chromophore or a light scattering particle and signal generation is external to and independent of the surface characteristics of the porous support. In these assays, sample flows through the device with a very limited contact time with the capture element of the device. Thus, sensitivity of the assay is limited by the capture efficiency of the system. Many of these devices suffer from highly variable flow rates as minor changes in the sample composition occur.
The devices of the current invention allow the sample incubation to occur over a period of time to improve capture efficiency but also minimize the user intervention required to complete the assay. The devices also provide an increase in assay performance by allowing all available sample to flow across the optical member and through channels within the optical member. Because the contact time of sample with the test surface is controlled, the devices are less sensitive to variable flow rates than other prior art devices. Also in the devices of this invention, the signal generation is inherent in the composition and construction of the flow through support. Drying of the optical surface from below instead of from above decreases the risk of damaging the optical surface prior to the detection step.
To this end, an aspect of the present invention involves an optical assay device for the detection of an analyte of interest that conveniently allows the device, not the user, to control the flow rate and mass transport of a sample, i.e., any fluid medium, gas or liquid, through the device. The optical assay device includes a base having an absorbent material, and a member having an optically active test membrane or stack that is rotatably coupled to the base for rotation between a lowered position and a raised position. The optically active test stack includes all of the components necessary to generate the optical signal on the test surface including the capture reagent and to allow for sample flow. In the lowered position, the optically active test stack contacts the absorbent material for drawing the sample across the optical member and through channels within or around the optical member. In the raised position, the optically active test stack does not contact the absorbent material and allows for increased sample contact time with optical test surface.
This simple control feature improves analyte capture efficiency by increase sample contact time with the capture reagent and for rapid fluid flow. The control feature is a simple manually operated rotation of the device that minimizes user interaction while allowing for the execution of a number of assay manipulations.
In a preferred embodiment of the present invention, the optical assay device may include any or all of the following:
the member is rotatably coupled to the base through a cam mechanism, the cam mechanism including at least one ramp, whereby the member moves up at least one ramp when the member is moved from the lowered position to the raised position, and down at least one ramp when the member is moved from the raised position to the lowered position;
the optical assay device also includes a retaining mechanism for retaining the member to the base;
the optical assay device also includes a stop mechanism for restraining the rotation of the member to the lowered position, the raised position, and therebetween;
the member includes a projection adapted to be manipulated by a user""s fingers to assist in rotating the member;
the base includes a pair of finger grips to assist in holding the base;
the optically active test stack includes an optically functional layer made of an amorphous silicon or other material to make the test surface reflective and having a thickness between 1000 and 5000 xc3x85;
a support carries the optically active test stack, the support preferably made of nylon, track-etch polycarbonate, nitrocellulose, or polysulfone;
the optically functional layer is coated with an antireflective layer having a thickness between 400 and 700 xc3x85; and
the antireflective layer is coated with an attachment layer made of a diamond-like carbon (alternatives to diamond-like carbon include thin layers of Ni, Ge, or polymers like siloxones or film forming latexes) having a thickness between 50 and 1000 xc3x85.
Another aspect of the present invention involves an optical assay device that includes a base having absorbent material, and a member including an optically active test stack. The base lies generally in a first plane, and the member lies generally in a second plane that is parallel to the first plane. The member is operatively associated with the base for movement between a lowered position and a raised position. In the lowered position, the optically active test stack contacts the absorbent material and the member lies generally in the same plane as the base for drawing a sample through the stack. In the raised position, the optically active test stack does not contact the absorbent material and the member does not lie in the same plane as the base.
If sample is applied with the member in the lowered position, flow will initiate immediately. This is advantageous in an assay system where extremely high sensitivity is not required. The sample will flow until exhausted and then wash can be directly applied to the member in the lowered position. Additional reagents can be applied to the member in the lowered position until the assay is complete. An alternative would be to add a reagent, preferably the amplification reagent, to the member in the raised position. In this case, the amplification reagent will incubate on the optically active surface until the member is moved into the lowered position for removal of the amplification reagent and a final wash prior to read.
In assay systems where sensitivity is a requirement, the sample should be applied with the member in the raised position to allow for efficient capture of the available analyte. After the incubation period, the sample flow is initiated by moving the member to the lowered position. The member will remain in the lowered position until the wash step is complete. Then the member will be moved to the raised position for the addition of other reagents. The member remains in the raised position until the incubation period is complete and then is moved to the lowered position to remove reagent and wash the test surface. If necessary the reagent cycle could be repeated until the assay is complete.
A further aspect of the present invention involves an optical assay device for the detection of an analyte of interest that includes a base having absorbent material, and a generally circular member including a central axis. The generally circular member includes a central aperture and an optically active test stack that covers the aperture. The generally circular member is rotatably coupled to the base through a cam mechanism for rotation about the axis between a lowered position and a raised position. In the lowered position, the optically active test stack contacts the absorbent material for drawing a sample through the stack. In the raised position, the optically active test stack does not contact the absorbent material. The optical assay device further includes a stop mechanism for restraining rotation of the generally circular member between the lowered position and the raised position, and a retaining mechanism for retaining the generally circular member to the base.
In a preferred embodiment of the aspect immediately described above, the cam mechanism includes a plurality of ramping members extending from the base, and a plurality of respective ramping members extending from the generally circular upper member that are adapted to slidably cooperate with the ramping members upon rotation of the generally circular member for raising and lowering the generally circular member; and
the base includes a well that carries the absorbent material.
Another aspect of the present invention includes an optical assay device including a base having absorbent material, and a member including an optically active test stack. The device further includes means for raising and lowering the member between a lowered position and a raised position. In the lowered position, the optically active test stack contacts the absorbent material for drawing a sample through the surface. In the raised position, the optically active test stack does not contact the absorbent material.
In a preferred embodiment of the aspect immediately described above, the optical assay device includes means for retaining the member to the base.
A still further aspect of the present invention involves a method for detecting an analyte of interest in a test sample. The method includes providing an optical assay device, the optical assay device comprising a base including absorbent material, and a member including an optically active test stack, the member rotatably coupled to the base for rotation between a lowered position where the optically active test stack contacts the absorbent material and a raised position where the optically active test stack does not contact the absorbent material; providing the optical assay device in the lowered position where the optically active test stack contacts the absorbent material for drawing a sample through the stack; applying the test sample to the optically active test stack; applying a conjugate to the optically active test stack; applying a wash to the optically active test stack; rotating the member to the raised position where the optically active test stack does not contact the absorbent material; applying an amplifying reagent in solution to the optically active test stack; rotating the member to the lowered position so that the solution containing the amplifying reagent is drawn through the optically active test stack, thereby depositing the amplifying reagent; and observing the optically active test stack for a visual indication of the presence of the analyte of interest.
Other features and advantages of the inventions are set forth in the following detailed description and drawings, which are intended to illustrate, but not limit, the invention.